The purpose of this grant is to continue my training to enable me to become an independent investigator using molecular biology techniques to study cardiovascular diseases. The scientific goal of this grant is to isolate and characterize the macrophage nitric oxide synthase gene and to study its regulation. Nitric oxide (NO) is a novel second messenger with a variety of functions: NO is released by endothelial cells to induce smooth muscle relaxation, serving as a vasodilator; NO is released by neurons to stimulate guanylate cyclase in adjacent neurons, serving as a neurotransmitter; and NO is released by macrophages to kill bacteria, fungi, parasites, and tumor cells, serving as a cytotoxic agent. NO is produced by nitric oxide synthase (NOS). The NOS found in brain has been purified recently in this laboratory, but it appears to be distinct from forms of NOS found in other tissues. The versatility of NO suggests that defects or mis-regulation of NOS could play a role in a variety of diseases. Decreased production of endothelial NO could lead to reduced smooth muscle relaxation and thus to hypertension. Bacterial induction of endothelial and macrophage NOS and the subsequent over-production of NO might cause septic shock. Impaired NO production in macrophages may be a factor in a rare set of diseases predisposing to infection since bacterial killing is deficient. The specific aims of this project are as follows. The Mac-NOS gene will be isolated and sequenced. The Mac-NOS cDNA will be expressed in tissue culture cells, and the enzymology of the purified product of the Mac-NOS cDNA will be characterized. Expression of Mac-NOS mRNA and protein will be localized histologically, and expression of other homologous genes localized as well. In addition the genomic structure of the Mac-NOS gene including its promoter region will be characterized. Furthermore a novel assay will be used to search for transcriptional regulators of the Mac-NOS gene.